7-aminodeacetoxycephalosporanic acid (7-ADCA) is one of the important intermediates for the production of cephalosporins cefalexin, cefradine, and cefadroxil which are antibiotic compounds commonly and long used in humans and animals. The industrial process for synthesizing 7-ADCA mainly includes two steps: a chemical ring expansion of penicillin G to phenylacetyl-7-ADCA and an enzymatic side chain cleavage of phenylacetyl-7-ADCA. However, the chemical reaction of the ring expansion is complex and expensive, and the by-products and the organic solvents (such as pyridine and HBr) are toxic to the environment. Therefore, an enzymatic reaction is greatly desirable to replace such chemical reaction.
It has been reported that a natural enzyme, deacetoxycephalosporin C synthase (DAOCS, or expandase), may be responsible for the catalysis of the expansion reaction. Streptomyces sp. (such as Streptomyces clavuligerus, Streptomyces ambofaciens and Streptomyces chartreusis) can produce expandase. As illustrated in EP-A-0341892, expandase could be obtained from Streptomyces clavuligerus, and has been cloned. Expandase has been well studied for its chemical and functional properties, see EP-A-0366354. Unfortunately, the native expandase has less substrate specificity to penicillin G than the normal substrate penicillin N (Rollins, M. J. et al., Can. J. Microbiol. 34: 1196-1202, 1988 and Maeda, K. et al., Enzyme and Microbial Technology, 17: 231-234, 1995). Penicillin G is commercially available at a low cost. In contrast, penicillin N is expensive and not easily available. Furthermore, even though penicillin N is expanded, its side chain cannot be easily removed. Accordingly, the chemical synthesis of 7-ADCA, rather than an enzymatical synthesis, is still used in industrial production.
There are a number of prior art references on the production of 7-ADCA with expandase. U.S. Pat. No. 5,731,165 describes a process for the preparation and recovery of 7-ADCA via enzymatic ring expansion activity on penicillin G, with a Penicillium chrysogenum transformant strain expressing expandase. U.S. Pat. No. 5,559,005 discloses a bioprocess for preparing 7-amino-cephalosporanic acid (7-ACA) or 7-ADCA with a transformed stain of Penicillium chrysogenum having expandase activity wherein adipoyl-6-amino-penicillanic acid (adipoyl-6-APA) is used as a substrate. Nevertheless, since both adipoyl-6-APA and penicillin G are poor substrates for native expandase in vitro (U.S. Pat. No. 5,559,005), a native expandase is not optimistic to have a high expansion efficiency while applied in vivo.
Recently, Chin H. S. et al. (Biochemical and Biophysical Research Communications, 287: 507-513, 2001) discloses a mutated DAOCS comprising an amino acid substitution of N304L. U.S. Pat. No. 5,919,680 describes a mutated expandase which has an altered amino acid sequence from a native expandase, resulting in an altered substrate specificity. Several amino acid positions have been mentioned in that patent, and the mutated expandase created by changing one or more of the mentioned amino acids shows a higher activity ratio of penicillin G to penicillin N in a mixture of these two substrates, but has a lower activity on penicillin G and penicillin N individually than wild-type expandase. Therefore, there is still a need to develop a mutated penicillin expandase having more substrate specificity and enzymatic activity on penicillin G.